Covalent binding assay

The toxicologist can still use of in silico technology36 to assess the chemical-based toxicity potential of various structural series and many molecular methodologies can still be applied, including covalent binding assays, mitochondrial assays and oxidative stress assays.29-37... 

Cy Reactive Metabolite Screening and Covalent-Binding Assays... 

A second issue is related to the fact that the rate and quantitative importance of the metabolic pathway(s) that leads to the generation of chemically reactive intermediates may, or may not, correlate with the overall metabolic turnover of the compound in the in vitro covalent binding assay systems. Thus, it is not advisable to... 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Additional assays used in early pharmaceutical property profiles usually include plasma protein binding, individual cytochrome P450 assays, stability in the presence of serum, production of metabolites likely to be involved in covalent binding to biomolecules, and interaction with efflux pumps ... 

Fig. 2.17 Relative non-covalent binding affinities of a variety of drug candidates to RCS4 and G alpha proteins based on the relative S/N ratios in the CPC spin column/ESI-MS assays to that of 250 pg of the respective compound (normal font) and after normalizing all the values...

Mrksich and co-workers developed a MALDI-based assay scheme making use of a target surface modification by self-assembled monolayers (SAMs) [22]. This combination of SAMs and MALDI is predominantly called SAMDI (selfassembled monolayers for MALDI). For SAMDI, a self-assembled monolayer with reactive end groups is used in order to covalently bind enzyme substrates to a surface. To... 

DEHA was not mutagenic or genotoxic in a variety of assays, nor did it covalently bind to DNA in vivo 3"... 

In male and female rats exposed to 10, 50, 250, or 12 50 ppm vinyl bromide in a lifetime inhalation study, there was a dose-related increase in angiosarcomas of the liver in both sexes. A significant increase in hepatocellular neoplasms was also seen in male rats exposed at 250ppm and in female rats exposed at 10, 50, and 250ppm. The lack of increase in hepatocellular neoplasms in rats at the 12 50 ppm level was probably due to their early mortality and termination at 72 weeks. In limited mice studies, no local tumors were produced by skin application or subcutaneous administration. Vinyl bromide is mutagenic in bacterial assays and Drosophilas It is activated via a P-450-dependent pathway to its epoxide that can covalently bind to DNA. ... 

From a purely pragmatic perspective, it is clear that reactive metabolites are linked with toxicity and that a circumstantial link can be made to idiosyncratic toxicides. Consequently, even though the mechanism of this toxicity is not fully understood, since assays are available to measure the potential for bioactivation in an ideal world one would not carry this liability forward. Conversely, it is not an ideal world, all drug molecules have challenges and the definition of therapeutic index (i.e., the ratio between the toxic exposure and the therapeutic exposure) is critical. Covalent binding of reactive metabolites to macromolecules is a crude measure and not a full predictor of toxicity and it is well known that toxicity can be ameliorated by a lower dose. Furthermore, the so-called definitive assays require radiolabeled drug material which is expensive and generally slow to produce. 

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